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IFN-gamma regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells.

ABSTRACT: In this study we examined the ability of interferon-gamma (IFN-gamma) to regulate mammary epithelial cell growth and gene expression, with particular emphasis on two genes: Maspin (a member of serine protease inhibitor superfamily), and the lysosomal aspartyl endopeptidase cathepsin D (CatD). The protein products of these genes are critically involved in regulation of multitude of biological functions in different stages of mammary tissue development and remodeling. In addition, the expression of Maspin is down-regulated in primary breast cancer and is lost in metastatic disease, while CatD is excessively produced and aberrantly secreted by breast cancer cells. We report that IFN-gamma receptors are expressed in mammary epithelial cells, and receptor engagement by IFN-gamma transduces the IFN-gamma signal via Stat-1 resulting in decreased vacuolar pH. This change in vacuolar pH alters CatD protein processing and secretion concurrent with increased Maspin secretion. In addition, IFN-gamma exerts a suppressive effect on cell growth and proliferation, and induces morphological changes in mammary epithelial cells. Our studies also reveal that breast cancer cells, which are devoid of Maspin, are refractory to IFN-gamma with respect to changes in vacuolar pH and CatD. However, Maspin transfection of breast cancer cells partially sensitizes the cells to IFN-gamma's effect, thus providing new therapeutic implications.

SUBMITTER: Khalkhali-Ellis Z

PROVIDER: S-EPMC2575810 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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IFN-gamma regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells.

Khalkhali-Ellis Zhila Z Abbott Daniel E DE Bailey Caleb M CM Goossens William W Margaryan Naira V NV Gluck Stephen L SL Reuveni Moshe M Hendrix Mary J C MJ

Journal of cellular biochemistry 20080901 1

In this study we examined the ability of interferon-gamma (IFN-gamma) to regulate mammary epithelial cell growth and gene expression, with particular emphasis on two genes: Maspin (a member of serine protease inhibitor superfamily), and the lysosomal aspartyl endopeptidase cathepsin D (CatD). The protein products of these genes are critically involved in regulation of multitude of biological functions in different stages of mammary tissue development and remodeling. In addition, the expression o ...[more]

PMID: 18494001

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Project description:BackgroundIFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown.MethodsIn this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry.ResultsThe targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control.ConclusionsThese results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Project description:Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A1 or knockdown of Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland.Abbreviations: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; shNT: short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.

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IFN-gamma regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells.

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IFN-gamma regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells.

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